Compositions and Methods for Analyzing Modified Nucleotides

Abstract

A method for identifying any of the presence, location and phasing of modified cytosines (C) in long stretches of nucleic acids is provided. In some embodiments, the method may comprise (a) reacting a first portion of a nucleic acid sample containing at least one C and/or at least one modified C with a DNA glucosyltransferase and a cytidine deaminase to produce a first product and/or reacting a second portion of the sample with a dioxygenase, optionally a DNA glucosyltransferase and a cytidine deaminase to produce a second product and; (b) comparing the sequences from the first and optionally the second product obtained in (a), or amplification products thereof, with each other and/or an untreated reference sequence to determine which Cs in the initial nucleic acid fragment are modified. A modified TET methylcytosine dioxygenase with improved efficiency compared to unmodified TET2 at converting methylcytosine to carboxymethylcytosine is also provided.

What is claimed is: 
     
         1 .- 8 . (canceled) 
     
     
         9 . A method comprising:
 a. determining the location of substantially all modified cytosines in a test nucleic acid fragment having a length of at least 2 kb, to obtain a pattern of cytosine modification;   b. comparing the pattern of cytosine modification in the test nucleic acid fragment with the pattern of cytosine modification in a reference nucleic acid fragment; and   c. identifying a difference in the pattern of cytosine modification in the test nucleic acid fragment in cis relative to the reference nucleic acid fragment.   
     
     
         10 . The method of  claim 9 , wherein the method comprises comparing the pattern of cytosine modification for the test nucleic acid fragment, wherein the test nucleic acid is linked, in cis, to a gene in a transcriptionally active state to the pattern of cytosine modifications in the same intact nucleic acid fragment that is linked, in cis, to the same gene in a transcriptionally inactive state. 
     
     
         11 . The method of  claim 9 , wherein transcription of the gene is correlated with a disease or condition. 
     
     
         12 . The method of  claim 9 , wherein the method comprises comparing the pattern of cytosine modification for a nucleic acid fragment from a patient that has a disease or condition with the pattern of cytosine modification in the same nucleic acid fragment from a patient that does not have the disease or condition. 
     
     
         13 . The method of  claim 9 , wherein the method comprises comparing the pattern of cytosine modification or lack of modification for a nucleic acid fragment from a patient is undergoing a treatment with the pattern of cytosine modification or lack of modification in the same intact nucleic acid fragment from a patient that has not been treated with the agent. 
     
     
         14 . A method according to  claim 9 , wherein the difference in the pattern of cytosine modification in the test nucleic acid fragment relative to the reference nucleic acid fragment corresponds to a variant single nucleotide polymorphism, an insertion/deletion or a somatic mutation associated with a pathology. 
     
     
         15 . A method according to  claim 9 , wherein identifying a difference in the pattern of cytosine modification in the test nucleic acid fragment relative to the reference nucleic acid fragment further comprises identifying a difference in the pattern of unmodified cytosine in cis or a difference in the pattern of modified cytosines in cis.